A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris.

As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45°C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn(2+), Cu(2+), and Fe(2+) and promoted by Co(2+) and Ca(2+). Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics ampicillin and kanamycin against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL production, the characterization, and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.

Novel RNA-binding protein P311 binds eukaryotic translation initiation factor 3 subunit b (eIF3b) to promote translation of transforming growth factor ß1-3 (TGF-ß1-3).

P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-ß1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 µm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-ß1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-ß 5'UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-ßs 5'UTRs and eIF3b, stimulates the translation of TGF-ß1, 2, and 3.

In pulmonary lymphangioleiomyomatosis expression of progesterone receptor is frequently higher than that of estrogen receptor.

Lymphangioleiomyomatosis (LAM) of the lung is a rare low-grade malignancy affecting primarily women of childbearing age. LAM is characterized by the proliferation of SMA and HMB-45 positive spindle-shaped and epithelioid cells throughout the lung in the form of discrete lesions causing cystic destruction and ultimately respiratory insufficiency. LAM occurs sporadically or in patients with tuberous sclerosis complex (TSC) and is etiologically linked to mutations in the TSC1 and TSC2 genes. Although LAM cells are known to express estrogen and progesterone receptors (ER and PR, respectively), their respective expression level was never determined. Therefore, here we measured the immunohistochemical expression of ERs and PRs in a large series of pulmonary LAM cases using the Aperio Spectrum Analysis Platform. Our case series comprised open lung biopsy specimens from 20 LAM patients and lungs explanted during the course of lung transplant from 24 patients. All cases were positive for ER and PR. PR expression was statistically significantly higher than ER in 80 % of the biopsies while ER predominated only in one case. Specimens from explanted cases of LAM had relatively fewer PR-positive nuclei. As a result, PR expression was significantly higher than ER in 38 % of the cases, whereas ER predominated in 33 %. Overall, PR expression predominated in 57 % of cases and ER in 21 %. These data indicate that PR frequently prevails over ER in pulmonary LAM. LAM is unusual in its high PR/ER ratio; other female neoplasms show a definite prevalence of ER. Our findings therefore warrant further study of PR function in LAM.